WO2018181679A1 - Method for producing peptide - Google Patents

Abstract

The present invention addresses the problem of providing a novel peptide solid-phase synthesis method for synthesizing a peptide in a large quantity. The present invention also addresses the problem of providing a novel peptide solid-phase synthesis method for synthesizing a long-chain peptide having high purity. The present invention also addresses the problem of providing a novel peptide solid-phase synthesis method which causes few side reactions. The present invention relates to a method for producing a peptide, characterized in that the peptide is solid-phase-synthesized while stirring with a centrifugal stirrer equipped with no impeller.

Description

Method for producing peptide

The present invention relates to a method for producing a peptide. More specifically, the present invention relates to a method for mass synthesis of long-chain peptides by solid phase synthesis.

Shaking stirring, nitrogen bubbling stirring, stirrer stirring and the like are known as stirring means usually used in the solid phase synthesis method of long chain peptides.
On the other hand, centrifugal agitation bodies are known (Patent Documents 1 to 4 etc.) and there are examples applied as parts of liposome production apparatuses (Patent Document 5), but centrifugal agitation bodies are used in the field of organic synthesis. There has been no report of an example of application as a component of a peptide solid-phase synthesizer.

WO2010 / 150656 pamphlet JP 2014-124540 A Japanese Patent Laying-Open No. 2015-171695 JP 2015-47540 A JP 2016-1117005 A

The present invention includes a novel peptide solid-phase synthesis method for synthesizing a large amount of peptide, a novel peptide solid-phase synthesis method for synthesizing a long-chain peptide with high purity, and a novel peptide solid-phase synthesis method with few side reactions. The object is to provide one or more solid phase synthesis methods.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that high-purity long-chain peptides can be synthesized in large quantities by applying a centrifugal stirring body to a peptide solid phase synthesis method. Based on the findings, further research has been made and the present invention has been completed.

That is, the present invention relates to the following inventions.
[1] A method for producing a peptide, characterized in that the peptide is solid-phase synthesized under stirring of a centrifugal stirring body without blades.
[2] A centrifugal stirring body without blades
A main body that rotates about a rotation axis;
An inlet provided on the surface of the body;
A discharge port provided on the surface of the main body;
A flow path connecting the suction port and the discharge port,
The suction port is disposed at a position closer to the rotation shaft than the discharge port,
The manufacturing method according to [1], wherein the discharge port is a stirring rotator that is disposed at a position on the outer side in the centrifugal direction from the rotation shaft than the suction port.
[3] Use of a centrifugal stirring body without a blade for peptide synthesis by a solid phase method.
[4] A reaction vessel for peptide solid phase synthesis equipped with a centrifugal stirring body without blades.
[5] The reaction vessel for peptide solid phase synthesis according to [4], comprising a glass filter.

The present invention relates to a novel peptide solid-phase synthesis method for synthesizing a large amount of peptide, a novel peptide solid-phase synthesis method for synthesizing a high-purity long-chain peptide, and a side reaction (for example, a fragment peptide resin cleavage reaction). In addition, one or more solid phase synthesis methods can be provided among the novel peptide solid phase synthesis methods with less undesirable side chain deprotection reactions before the final coupling reaction.
The reaction in the solid phase synthesis method includes a protective group introduction reaction before the coupling reaction, a carboxyl group or amino group activation reaction involved in the peptide coupling reaction before the coupling reaction, a coupling reaction, and a resin cleavage. Reactions such as a deprotection reaction after a reaction, a coupling reaction, and a resin cleavage reaction are included.

It is a structural example of the container carrying the centrifugal stirring body without a blade | wing. (A) It is a top view of the rotary body for stirring which concerns on embodiment of this invention. (B) It is a front view of the rotating body for stirring (citing FIG. 1 of the patent 4418019 gazette). (A) It is the top view which showed the action | operation of the rotary body for stirring. (B) It is the front view which showed the action | operation of the rotary body for stirring (citing FIG. 2 of patent 4418019). It is a perspective view which shows one aspect | mode of the stirrer main body (quoting FIG. 2 of Unexamined-Japanese-Patent No. 2014-124540, and there is a part change). It is a perspective view which shows one aspect | mode of the main body of a stirrer (refer FIG. 4 of Unexamined-Japanese-Patent No. 2014-124540, and there exists a part change). It is a perspective view which shows one aspect | mode of the stirrer main body (quoting FIG. 6 of Unexamined-Japanese-Patent No. 2014-124540, and there is a part change). FIG. 2 is a front view (side view) showing one embodiment of a stirring device (quoting FIG. 1 of JP-A-2015-171695, with some modifications). The HPLC chromatogram of the product of Comparative Example 1 is shown. The HPLC chromatogram of the product of Comparative Example 2 is shown. The HPLC chromatogram of the product of Experimental Example 3 is shown. The HPLC chromatogram of the acetonitrile solution of the product of Experimental example 5 is shown. The HPLC chromatogram of the product of Comparative Example 3 is shown. The HPLC chromatogram of the product of Experimental Example 6 (synthetic product using M-Revo after the fifth residue coupling step) is shown. The HPLC chromatogram of the product of Experimental example 6 (shaking machine synthetic | combination product) is shown. The HPLC chromatogram of the product of Experimental Example 7 is shown. The HPLC chromatogram of the product of Experimental Example 8 is shown. The HPLC chromatogram of the product of Comparative Example 4 is shown. The HPLC chromatogram of the product of Comparative Example 5 is shown. The HPLC chromatogram of the product of Experimental Example 9 is shown. The HPLC chromatogram of the product of Comparative Example 6 is shown.

The present invention provides a method for producing a peptide, characterized in that the peptide is solid-phase synthesized under stirring of a centrifugal stirring body having no blades.

Centrifugal stirring body without blades A centrifugal stirring body without blades refers to, for example, a stirring body that has no blades in appearance and has a function of stirring fluid using centrifugal force. The fluid preferably contains a resin, a target peptide constituent amino acid, or a combination thereof. The structure of the centrifugal agitator without blades may be, for example, a rotating body that forms a flow path by connecting a suction port close to the rotation axis and a discharge port far from the rotation axis. The principle of agitation is, for example, that centrifugal force acts in the flow path due to the rotation of the stirring body, fluid is discharged in the lateral direction, the vertical flow path becomes negative pressure, and a negative pressure suction flow is generated below. There may be.

An example of the configuration of a container equipped with a centrifugal stirring body without blades is shown in FIG. When a centrifugal stirring body without blades is rotated, centrifugal force is generated at the discharge port (a), and fluid is discharged from (a) in the lateral direction. Accordingly, a suction force is generated at the suction port (b), and a tornado-like vortex flow (c) is generated. The longitudinal flow path becomes negative pressure and a negative pressure suction flow is generated in the downward direction, and a “push → pull” flow is generated. A pulsation (pulse) is transmitted from the centrifugal stirring body without blades to the stirring flow, and the stirring flow is spread throughout.
As the centrifugal stirring body without blades, M-Revo (registered trademark), E-REVO, etc., manufactured by Medec Co., Ltd. can be used.

As the centrifugal stirring body without blades, for example, the stirring body described in WO2010 / 150656 pamphlet, Japanese Patent No. 4418019, Japanese Patent Application Laid-Open No. 2014-124540, or the like may be used.
That is, the centrifugal stirring body without blades is
(1) a main body that rotates about a rotation axis;
An inlet provided on the surface of the body;
A discharge port provided on the surface of the main body;
A flow path connecting the suction port and the discharge port,
The suction port is disposed at a position closer to the rotation shaft than the discharge port,
The discharge port may be a rotating body for stirring (see, for example, FIGS. 2 and 3), characterized in that the discharge port is disposed at a position on the outer side in the centrifugal direction from the rotation shaft with respect to the suction port.
(2) a main body having a circular cross section perpendicular to the rotation axis direction;
An inlet provided on the surface of the body;
A discharge port provided on the surface of the main body;
A flow path connecting the suction port and the discharge port,
The suction port is disposed at a position closer to the rotation shaft than the discharge port,
The discharge port may be a stirring rotator (see, for example, FIGS. 2 and 3), characterized in that the discharge port is disposed at a position radially outward from the rotation shaft with respect to the suction port, or ,
(3) A stirrer main body that rotates a cylindrical rotating member formed of a cylindrical housing whose upper end is closed by a top plate around a central axis of the cylindrical housing by a rotation drive shaft fixed to the top plate. ,
The cylindrical rotating member is
A plurality of discharge openings formed in the peripheral surface of the cylindrical housing;
A plurality of extrusion protruding plate portions provided so as to protrude inwardly on the inner peripheral surface of the cylindrical housing;
A suction opening provided at the lower end of the cylindrical housing;
When the cylindrical rotating member is rotated, a part of the stirring liquid that forms the internal circulation flow is generated by causing the internal stirring flow to circulate the existing stirring liquid around the central axis line by the extrusion protruding plate portion. A stirrer main body (for example, FIGS. 4 to 6) characterized in that it is discharged from the discharge opening to the outside as an external discharge flow from the discharge opening by centrifugal force and external stirring liquid is taken into the inside as a suction flow from the suction opening. For example).

As a stirring apparatus for stirring conditions, a container equipped with the stirring body can be used. “Installation” refers to incorporation as a part of the apparatus.
As a stirring device,
Comprising a rotating body for stirring and a flow resistor disposed adjacent to each other;
The stirring rotating body includes:
A main body that rotates about a rotation axis;
An inlet provided on the surface of the body;
A discharge port provided at a position on the outer surface of the main body in the centrifugal direction from the rotation shaft with respect to the suction port;
A flow path connecting the suction port and the discharge port,
The flow resistor is
A resistor body that rotates about a resistor rotation axis;
A resistor inlet provided on the surface of the resistor body;
A resistor discharge port provided at a position on the outer surface of the resistor main body in the centrifugal direction from the resistor rotation shaft with respect to the resistor suction port;
A resistor flow path connecting the resistor suction port and the resistor discharge port,
The resistor body is configured to have a shape or a size different from that of the main body of the stirring rotator, or is arranged in a posture different from that of the main body of the stirring rotator. A stirrer (see, for example, FIG. 7) may be used (see JP-A-2015-171695).
The agitator described above is useful as a reaction vessel for peptide solid phase synthesis.
Moreover, it is preferable that the reaction vessel for peptide solid phase synthesis is equipped with a cylindrical vessel and a centrifugal stirring body without blades (see, for example, FIG. 1). The cylindrical container may have a bottom surface and a side surface made of plastic or glass. The cylindrical container may or may not have a plastic or glass lid on the top. If peptide solid-phase synthesis can be carried out in the container, the centrifugal stirring body without blades may be arranged at any position of the cylindrical container, but it may be arranged at the lower center of the cylindrical container. preferable. Reactants, reaction liquid and / or washing liquid can be put from the upper part of the cylindrical container.
Further, the peptide solid phase synthesis reaction vessel preferably further includes a heat medium jacket (cover), a heat medium suction port, and a heat medium discharge port mounted on the outside of the cylindrical container (for example, see FIG. 1). reference). For example, circulating water at about 5 to 80 ° C. may be put into the heating medium jacket from the heating medium suction port, and the circulating water may be discharged from the heating medium discharge port via the circulating water jacket.
The peptide solid phase synthesis reaction vessel preferably further includes a glass filter. The glass filter may be, for example, a crucible type, a buch funnel type, or a plate shape, and can be obtained, for example, by purchasing a commercially available product. The plate diameter of the glass filter is not particularly limited and can be changed as appropriate. The size of the pores of the glass filter may be smaller than the resin (solid phase carrier) used for solid phase synthesis, and may be a size capable of filtering the liquid while the resin is held on the filter. The glass filter is preferably disposed above the bottom surface of the reaction vessel for peptide solid phase synthesis and / or below the centrifugal stirring body without blades (see, for example, FIG. 1). By mounting the glass filter, the operation of separating the resin and the liquid such as the reaction solution and the washing solution can be carried out simply and quickly, and the operation efficiency of the peptide solid phase synthesis can be improved.
The reaction vessel for peptide solid phase synthesis is preferably equipped with a cock, and can be obtained, for example, by purchasing a commercially available product. The cock (stopper) is preferably arranged below the bottom surface of the reaction vessel for peptide solid phase synthesis and the glass filter (see, for example, FIG. 1). By installing a cock (stopper), a desired amount of liquid inside the reaction vessel for peptide solid phase synthesis can be poured out at any time, so that the operation of separating the resin from the liquid such as the reaction liquid and the washing liquid can be carried out more easily.
The reaction vessel for peptide solid phase synthesis is more preferably equipped with a glass filter and a cock.

The dimensions and centrifugal force of the rotor of the centrifugal stirring body are not particularly limited and can be appropriately changed.
The centrifugal stirring body preferably has a plurality of (for example, 2 to 10) discharge ports on the circumference. Is indicative of the stirring capacity of the rotor, the discharge amount coefficient (opening area total × circumferential length of the discharge port) is preferably 60cm ³ ~ 6000 cm ^3, more preferably 200cm ³ ~ 2000cm ^3.

The peptide peptide may be, for example, a peptide having 5 to 150 amino acid residues, a peptide having 5 to 34, a peptide having 15 to 100, or a peptide having 10 to 80. It may be 15 to 80 peptides, 10 to 60 peptides, or 15 to 60 peptides.
Peptides include, for example, Abarelix, Insulin and its analogs, Endothelin, β-Endorphin Oxytocin, Calcitonin, Carperitide, Glucagon, Glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), ghrelin, goserelin, cholecystokinin, sinapultide, atrial natriuretic peptide ( ANP), Secretin, Cetrorelix, Somatostatin, Degarelix, Desmopressin, Teduglutide, Teriparatide, Brain natriuretic peptide (BNP), asopressin , Parathormone, Bradykinin, Peginesatide, Lanreotide, β-Lipotropin, γ-Lipotropin, Leuprorelin, Linaclotide Alternatively, it may be a peptide such as Liraglutide or a salt thereof. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt. For example, pharmaceutically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts and the like can be mentioned. As acid addition salts, inorganic acid salts such as hydrochloride, nitrate, sulfate, phosphate; oxalate, acetate, trifluoroacetate, maleate, fumarate, tartrate, citrate, lactic acid Examples thereof include organic acid salts such as salts, malates, succinates, gluconates, ascorbates, and p-toluenesulfonic acid. Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; aluminum salt and zinc salt. Examples of ammonium salts include ammonium and tetramethylammonium salts. Examples of organic amine addition salts include addition salts such as piperidine. Of these, acid addition salts, organic acid salts and the like are preferable, and acetates are more preferable.

Since the process conditions of the solid-phase synthesis method have been well established in the past, there is no particular limitation other than using a centrifugal stirrer without a blade as the stirrer. Synthesis methods, etc.) can be employed. The reaction in the solid phase synthesis method includes a protective group introduction reaction before the coupling reaction, a carboxyl group or amino group activation reaction involved in the peptide coupling reaction before the coupling reaction, a coupling reaction, a resin cleavage reaction, a cup Reactions such as a deprotection reaction after a ring reaction and a resin cleavage reaction are included. DMF / 20% piperidine may be used for the de-Fmoc group (9-fluorenylmethyloxycarbonyl group) reaction. Trifluoroacetic acid may be used in the de-Boc group (tertiary butoxycarbonyl group) reaction. Further, the presence or absence of an unreacted amino group may be confirmed using a ninhydrin reaction by a method such as a Kaiser test.
See the Examples section for examples of solid phase synthesis methods.

Effect According to the method for producing a peptide of the present invention, for example, a large amount of peptide can be synthesized. The amount of peptide that can be synthesized is preferably larger than the amount of peptide synthesized using the method for producing a peptide without using a centrifugal stirring body without blades. For example, it may be 1 g or more, or 10 g or more. It may be 100 g or more, 200 g or more, or 300 g or more.
The peptide synthesized using the method for producing a peptide of the present invention preferably has a higher purity than a peptide synthesized using a method for producing a peptide that does not use a centrifugal stirring body without blades. A preferable HPLC purity is, for example, 60% or more, 70% or more, 80% or more, or 90% or more.
Peptides synthesized using the peptide production method of the present invention have fewer side-reaction products such as detrityl derivatives than peptides synthesized using a peptide production method that does not use a bladeless centrifugal stirring body. It is preferable. The detrityl content is preferably, for example, 5% or less, 3% or less, or 1% or less.

The present invention includes embodiments in which the above configurations are combined in various ways within the technical scope of the present invention as long as the effects of the present invention are exhibited.

EXAMPLES Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples at all, and many variations are within the technical idea of the present invention. This is possible by those with ordinary knowledge.

In the examples, the measurement was performed under the following HPLC conditions.
Column: Waters XBridge Shield18 3.5 μm 4.6 × 150 mm
Mobile phase A: 0.1% TFA aqueous solution Mobile phase B: 0.08% TFA acetonitrile solution Flow rate: 1 mL / min
Detector: UV 220 nm

In this disclosure, pseudo-pro is pseudoproline, Trt is trityl group, HOBt is 1-hydroxy-1H-benzotriazole monohydrate, DMF is N, N-dimethylformamide, DIC is diisopropylcarbodiimide, DIEA Is diisopropylethylamine, Oxyma is ethyl (hydroxyimino) cyanoacetate, TFA is trifluoroacetic acid, TIS is tri (isopropyl) silane, EDT is ethanedithiol, and Cleavage mixture is acetic acid / trifluoroethanol / dichloromethane (volume ratio) 10/10/80), IPE indicates isopropyl ether, and TFE indicates 2,2,2-trifluoroethanol.

Solid-phase synthesis of 34-residue peptide [Experiment 1] A-fragment-resin (Boc-Ser (tBu) -Val-Ser (pseudo-pro) -Glu (tBu) -Ile-Gln (Trt) -Leu-Met -His (Trt) -Asn (Trt) -Leu-Gly-O-Trt (2-Cl) -resin) (side chain protected AFR-resin) synthesis (using M-Revo (registered trademark))
1. Coupling reaction (1) H-Gly-O-Trt (2-Cl) -resin (80.00 g), Fmoc (9-Fluorenylmethoxycarbonyl) -amino acid (2.5 eq.), Activator HOBt (22.42 g, 2.5 eq.), Reaction solvent DMF (800 mL), DIC (25.70 mL) were added.
(2) Centrifugation was performed for 2 hours or more using M-Revo (registered trademark).
(3) The reaction solvent was removed, and the Fmoc-amino acid introduced resin was washed with DMF (800 mL), dichloromethane (800 mL), and DMF (800 mL) using M-Revo (registered trademark).
(4) A small amount of Fmoc-amino acid-introduced resin was sampled, and it was confirmed that the resin was not colored using a Kaiser test. If the resin beads are colored by the Kaiser test, the operations (1) to (3) are repeated until no color develops.
2. Deprotection of Fmoc group (5) 20% piperidine / DMF (800 mL) was added to the obtained Fmoc-amino acid-introduced resin.
(6) The mixture was centrifuged for 20 minutes or more using M-Revo (registered trademark).
(7) The reaction solvent was removed and washed with M-Revo (registered trademark) (DMF 800 mL), dichloromethane (800 mL), DMF (800 mL).
(8) It was confirmed by the Kaiser test that the resin beads were colored.
(9) Operations (1) to (8) were carried out until the following side chain protected AFR-resin (230 g) was obtained. However, the serine (pseudoproline) residue was introduced as Fmoc-valine-serine (pseudoproline), which is a dipeptide whose N-terminus is protected with an Fmoc group.

[Experimental example 2] B-fragment-resin (H-Lys (Boc) -His (Trt) -Leu-Asn (Trt) -Ser (tBu) -Met-Glu (tBu) -Arg (Pbf) -Val-Glu (tBu) -Trp (Boc) -Leu-Arg (Pbf) -Lys (Boc) -Lys (Boc) -Leu-Gln (Trt) -Asp (tBu) -Val-His (Trt) -Asn (Trt)- Synthesis of Phe-O-Trt (2-Cl) -resin) (side chain protected BFR-resin) (using M-Revo (registered trademark))
1. Introduction of Fmoc-amino acid into resin (1) Cl-Trt (2-Cl) -resin (40.00 g) and Fmoc-Phe-OH (56.17 g) were added to a reaction vessel under an argon gas atmosphere.
(2) Dichloroethane (400 mL) and DIEA (25.25 mL) were added.
(3) Centrifugation was performed for 3 hours or more using M-Revo (registered trademark).
(4) The reaction solvent was removed, and DIEA (5.05 mL), methanol (40 mL), and dichloroethane were added in a volume that allowed the whole to be stirred.
(5) Centrifugation was performed for 1 hour using M-Revo (registered trademark) under an argon gas atmosphere.
(6) The reaction solvent was removed and washed with DMF (400 mL), dichloromethane (400 mL) and DMF (400 mL) using M-Revo (registered trademark).
(7) A small amount of Fmoc-Phe-O-Trt (2-Cl) -resin was sampled, and it was confirmed by a Kaiser test that the resin beads were not colored.
2. Deprotection of Fmoc group (8) To the obtained Fmoc-Phe-O-Trt (2-Cl) -resin, 20% piperidine / DMF (400 mL) was added.
(9) Centrifugation was performed for 20 minutes or more using M-Revo (registered trademark).
(10) The reaction solvent was removed and washed with DMF (400 mL), dichloromethane (400 mL) and DMF (400 mL) using M-Revo (registered trademark).
(11) It was confirmed by the Kaiser test that the resin beads were colored.
(12) H-Phe-O-Trt (2-Cl) -resin was obtained.

3. Coupling reaction (13) H-Phe-O-Trt (2-Cl) -resin obtained in (12), Fmoc-amino acid (2.5 eq.), HOBt (19.59 g, 2.5 eq.), DMF (10-20 v / w), DIC (22.45 mL, 2.5 eq.) Was added.
(14) Centrifugation was performed for 2 hours or more using M-Revo (registered trademark).
(15) The reaction solvent was removed, and the Fmoc-amino acid-introduced resin was washed with DMF, dichloromethane, and DMF using M-Revo (registered trademark).
(16) A small amount of Fmoc-amino acid-introduced resin was sampled, and it was confirmed by a Kaiser test that the resin beads were not colored. If the resin beads are colored by the Kaiser test, the operations (13) to (15) are repeated until the resin beads are not colored.
4). Deprotection of Fmoc group (17) 20% piperidine / DMF (400 mL) was added to the obtained Fmoc-amino acid-introduced resin.
(18) Centrifugation was performed for 20 minutes or more using M-Revo (registered trademark).
(19) The reaction solvent was removed and washed with DMF (400 mL), dichloromethane (400 mL), and DMF (400 mL) using M-Revo (registered trademark).
(20) It was confirmed by the Kaiser test that the resin beads were colored.
(21) Operations (13) to (20) were carried out until the following side chain protected BFR-resin (310 g) was obtained.

[Experimental Example 3] Cutting out protected peptide from side chain protected BFR-resin (using M-Revo (registered trademark))
(1) A degassed leakage mixture (800 mL) was added to a container to which side chain protected BFR-resin (80 g) was added.
(2) Centrifugation was performed for 2 hours using M-Revo (registered trademark).
(3) The reaction solution was filtered, washed three times with a leakage mixture (80 mL), and further washed with dichloromethane (160 mL).
(4) The filtrate was concentrated under reduced pressure at an external temperature of 25 ° C.
(5) Dichloromethane (160 mL) was added to the concentrated residue to dissolve the crystals.
(6) IPE (4000 mL) was added to the reaction vessel, and the solution (5) was added dropwise.
(7) After stirring at room temperature for 1 hour, the crystals were filtered under reduced pressure and washed using IPE (800 mL).
(8) Vacuum-dried at room temperature to obtain side chain protected B-fragment (67.95 g).

[Comparative Examples 1 and 2]
Side chain protected B-fragments were obtained by the same solid phase synthesis method as in Experimental Example 3 except that a stirrer was used instead of M-Revo (registered trademark).

[HPLC analysis (Comparative Examples 1 and 2 and Experimental Example 3)]
The results of HPLC analysis of the acetonitrile solutions of the products of Comparative Examples 1 and 2 and Experimental Example 3 are shown in Tables 5 to 7 and FIGS. Tables 5, 6 and 7 show the retention times of each product (detrityl by-product or B-fragment) in HPLC analysis of acetonitrile solutions of the products of Comparative Example 1, Comparative Example 2 and Experimental Example 3, respectively. The content rate is shown. 8, 9 and 10 show HPLC chromatograms (gradient program A) of acetonitrile solutions of the products of Comparative Example 1, Comparative Example 2 and Experimental Example 3, respectively.

Table 8 summarizes the results of Experimental Example 3 and Comparative Examples 1 and 2.

From the above results, the HPLC purity of the B-fragment of Experimental Example 3 is 69.2%, which is equivalent to that of Comparative Examples 1 and 2, whereas the content of detrityl compound produced as a by-product is 4.79%. It was found to be less than half that of 1 and 2. That is, the solid phase synthesis method using M-Revo (registered trademark) yielded a B-fragment having a higher purity, that is, less undesirable detrityl form than the solid phase synthesis method using a stirrer.

[Experimental Example 4] Production of 34-residue peptide The side chain protected AFR-resin obtained in Experimental Example 1 was treated in the same manner as in Experimental Example 3 to excise the side chain protected A-fragment from the resin, and its C-terminus. After activating the carboxyl group, the side chain protected B-fragment was reacted to synthesize a side chain protector of a 34-residue peptide, and this side chain protective group was deprotected to obtain the target peptide.

[Experimental Example 5] Side chain protected A-fragment-resin (Boc-Ser (tBu) -Val-Ser (pseudo-pro) -Glu (tBu) -Ile-Gln (Trt) -Leu-Met-His (Trt ) -Asn (Trt) -Leu-Gly-O-Trt (2-Cl) -resin) (side chain protected AFR-resin) (using M-Revo (registered trademark))

1. Introduction of Fmoc-amino acid into resin (1) Fmoc-Leu-OH (2.5 eq.), DMF, HOBt (2.5 eq.), And DIC (2.5 eq.) Were added to an eggplant flask and stirred for 30 minutes. Add the reaction solution in the eggplant flask to the reaction vessel containing H-Gly-O-Trt (2-Cl) -resin (80.00 g), and stir for 2 hours or more using M-Revo (registered trademark). It was. The reaction solvent was removed, and the resin was washed with DMF, dichloromethane, and DMF to obtain Fmoc-Leu-Gly-O-Trt (2-Cl) -resin.

2. Deprotection of Fmoc group (2) A 20% piperidine / DMF solution (10 v / w) was added to Fmoc-Leu-Gly-O-Trt (2-Cl) -resin. After stirring for 20 minutes using M-Revo, the reaction solvent was removed. Wash with DMF (10 v / w x 5 times), dichloromethane (10 v / w x 5 times), DMF (10 v / w x 5 times), and H-Leu-Gly-O-Trt (2-Cl) -I got a resin.

3. Coupling reaction (3) To H-Leu-Gly-O-Trt (2-Cl) -resin, Fmoc-Asn (Trt) -OH (2.5 eq.), HOBt (2.5 eq.), DIC (2.5 eq. ), DMF was added in a volume (about 10 v / w) that allowed stirring. After stirring for 2 hours or more using M-Revo (registered trademark), the reaction solvent was removed. Washing with DMF, dichloromethane and DMF gave Fmoc-Asn (Trt) -Leu-Gly-O-Trt (2-Cl) -resin.
(4) A 20% piperidine / DMF solution (10 v / w) was added to Fmoc-Asn (Trt) -Leu-Gly-O-Trt (2-Cl) -resin. After stirring for 20 minutes using M-Revo (registered trademark), the reaction solvent was removed. Washing with DMF, dichloromethane and DMF gave H-Asn (Trt) -Leu-Gly-O-Trt (2-Cl) -resin.
(5) Thereafter, the operations of (1) and (2) were repeated for the introduction of each Fmoc-amino acid and the deprotection of the Fmoc group. After introducing Fmoc-Met-OH, argon bubbling was performed for 10 minutes or more before starting stirring.
(6) After coupling Boc-Ser (tBu) -OH (2.5 eq.) To the 12th residue in the same manner as in (2), the reaction solvent was removed. After washing with MeOH and drying under reduced pressure, side chain protected AFR-resin (144.12 g) was obtained.

4). Cutting out the protected peptide from the resin and measuring the purity (7) Side chain protected AFR-resin obtained by the procedures of (6) (approx. 20-50 mg of a partly collected product) was added to acetic acid / dichloromethane (volume ratio acetic acid / Dichloromethane = 1/9) was added and stirred for 1-2 hours. The reaction solution was diluted with acetonitrile and analyzed by HPLC.
As a result, the purity of the target A-fragment was found to be 94.1% (t _R = 27.4), whereas the content of detrityl compound produced as a by-product was found to be 0.55% (t _R = 21.5). (See FIG. 11 and Table 9, Gradient program B).

[Comparative Example 3] Side chain protected A-fragment-resin (Boc-Ser (tBu) -Val-Ser (pseudo-pro) -Glu (tBu) -Ile-Gln (Trt) -Leu-Met-His (Trt ) -Asn (Trt) -Leu-Gly-O-Trt (2-Cl) -resin) (side chain protected AFR-resin) (Fully automated microwave peptide synthesizer (Biotage Japan Initiator + Alstra)) use)

1. Introduction of Fmoc-amino acid into resin (1) H-Gly-O-Trt (2-Cl) -resin (122 mg) and DMF (4.5 mL) were added to the reaction vessel and allowed to swell for 20 minutes. After filtration, Fmoc-Leu-OH 0.5M DMF solution (0.8 mL, 0.4 mmol, 4 eq.), DIC 0.5M DMF solution (0.8 mL), and HOBt 0.2M DMF solution (2.0 mL) were added. After reacting by microwave (MW) irradiation at 75 ° C. for 5 minutes, the reaction solvent was removed. Washing with DMF (18 mL) gave Fmoc-Leu-Gly-O-Trt (2-Cl) -resin.

2. Deprotection of Fmoc group (2) A 20% piperidine / DMF solution (4.5 mL) was added to Fmoc-Leu-Gly-O-Trt (2-Cl) -resin. After stirring for 3 minutes, the reaction solvent was removed and 20% piperidine / DMF solution (4.5 mL) was added. After stirring for 10 minutes, the reaction solvent was removed. Washing with DMF (18 mL) gave H-Leu-Gly-O-Trt (2-Cl) -resin.
3. Coupling reaction (3) F-moc-Asn (Trt) -OH 0.5M DMF solution (0.8 mL), DIC 0.5M DMF solution (0.8 mL) in H-Leu-Gly-O-Trt (2-Cl) -resin, HOBt 0.2M DMF solution (2.0 mL) was added. After reacting by MW irradiation at 75 ° C. for 5 minutes, the reaction solvent was removed. Washing with DMF (18 mL) gave Fmoc-Asn (Trt) -Leu-Gly-O-Trt (2-Cl) -resin.

(4) Thereafter, similarly, the deprotection and Fmoc amino acid coupling were repeated according to the methods of (2) and (3) (coupling of Fmoc-His (Trt) -OH was allowed to react at 50 ° C. for 10 minutes) . After coupling the last 12th residue of Boc-Ser (tBu) -OH (4.0 eq.) In the same manner as in (1), the reaction solvent was removed and washed with MeOH (13.5 mL) When dried under reduced pressure, side chain protected AFR-resin (350 mg) was obtained.

4). Extraction and purity measurement of protected peptide from resin (5) Acetic acid / TFE / dichloromethane (volume ratio acetic acid / TFE / dichloromethane = 1/1) to side chain protected AFR-resin (partially collected approximately 20-50 mg) / 9) was added and stirred for 2 hours. The reaction solution was diluted with acetonitrile and analyzed by HPLC. As a result, the purity of the target A-fragment was found to be 66.1% (t _R = 27.9), whereas the content of detrityl compound produced as a by-product was 7.5% (t _R = 22.1). (See FIG. 12 and Table 10, Gradient program B).

[Experimental Example 6] 15-residue peptide-resin (H-Arg (Pbf) -Val-Glu (tBu) -Trp (Boc) -Leu-Arg (Pbf) -Lys (Boc) -Lys (Boc) -Leu- Synthesis of Gln (Trt) -Asp (tBu) -Val-His (Trt) -Asn (Trt) -Phe-O-Trt (2-Cl) -resin) (The 用 い EYELA high-speed shaker (Cute Mixier) CM-1000 type or As One high-speed shaker ASCM-1) was used for stirring. The body resin was divided into two, and half of the amount was synthesized by a stirring operation using M-Revo (registered trademark) and the other half by a stirring operation using a shaker.

1. Introduction of Fmoc-amino acid into resin (1) Under argon atmosphere, Cl-Trt (2-Cl) -resin (4.00 g), Fmoc-Phe-OH (6.32 g), DIEA (2.79 mL), dichloroethane in a reaction vessel (28.0 mL) was added. After stirring at room temperature for 3.5 hours, the mixture was washed 5 times with dichloroethane (28.0 mL).
(2) MeOH (4.00 mL), DIEA (0.8 mL), and dichloroethane (28.0 mL) were added to the washed resin. After stirring for 20 minutes, the reaction solvent was removed. Washing with DMF, dichloromethane and DMF gave Fmoc-Phe-O-Trt (2-Cl) -resin.

2. Deprotection of Fmoc group (3) Add 20% piperidine / DMF solution (40.0 mL, 10 v / w) to Fmoc-Phe-O-Trt (2-Cl) -resin and stir for 20 minutes, then remove reaction solvent did. Wash with DMF (28.0 mL, 7 v / w x 5 times), dichloromethane (28.0 mL, 7 v / w x 5 times), DMF (28.0 mL, 7 v / w x 5 times), and H-Phe-O -Trt (2-Cl) -resin was obtained.

3. Coupling reaction (4) Fmoc-Asn (Trt) -OH (9.73 g, 16.3 mmol), HOBt (2.20 g), DIC (2.52 mL), DMF (40.0 mL) were added to the flask and stirred for 30 minutes. The solution was added to H-Phe-O-Trt (2-Cl) -resin, the inside of the flask was washed with DMF, the whole was stirred for 2 hours, and then the reaction solvent was removed.
(5) The resin was washed with DMF to obtain Fmoc-Asn (Trt) -Phe-O-Trt (2-Cl) -resin.

4). Deprotection of Fmoc group (6) Add 20% piperidine / DMF solution (40.0 mL) to Fmoc-Asn (Trt) -Phe-O-Trt (2-Cl) -resin and stir for 20 min. did. Washing with DMF, dichloromethane and DMF gave H-Asn (Trt) -Phe-O-Trt (2-Cl) -resin.

5). Coupling reaction (7) H-Asn (Trt) -Phe-O-Trt (2-Cl) -resin, Fmoc-His (Trt) -OH (1.01 g), HOBt (2.20 g), DIC (2.52 mL) ), And DMF (volume that can be stirred) were added and stirred for 2 hours or longer, and then the reaction solvent was removed.
(8) The resin was washed with DMF, dichloromethane, and DMF to obtain Fmoc-His (Trt) -Asn (Trt) -Phe-O-Trt (2-Cl) -resin.
(9) Thereafter, deprotection and coupling were repeated in the same manner as in (4) and (5).
(10) After coupling Fmoc-Arg (Pbf) -OH at the 15th residue, after deprotecting the Fmoc group according to the same method as in (3), the resin was washed with dichloromethane and MeOH and dried under reduced pressure. A 15-residue peptide-resin (10.7 g) was obtained.

6). Cleavage of protected peptide from resin and purity measurement (11) A part of 15-residue peptide-resin (about 20-50 mg) was collected, and acetic acid / dichloromethane (volume ratio acetic acid / dichloromethane = 1/1) was added to it. And stirred for 1-2 hours. The reaction solution was diluted with acetonitrile and analyzed by HPLC. Table 11 and Fig. 13 (Gradient Program A) show the results of the synthetic product using M-Revo (registered trademark) for a part of the process (after the 5th residue coupling step). The results are shown in Table 12 and FIG. 14 (gradient program A). As apparent from the results, the former showed better HPLC purity than the latter (HPLC purity: 93.1% synthetic product using M-Revo (registered trademark) after the fifth residue coupling step, shaker Synthetic product 89.4%).

[Experimental Example 7] 16-residue peptide-resin (H-Glu (tBu) -Arg (Pbf) -Val-Glu (tBu) -Trp (Boc) -Leu-Arg (Pbf) -Lys (Boc) -Lys ( Boc) -Leu-Gln (Trt) -Asp (tBu) -Val-His (Trt) -Asn (Trt) -Phe-O-Trt (2-Cl) -resin) (fully automated microwave peptide synthesizer) (Uses Biotage Japan Initiator + Alstra)

1. Introduction of Fmoc-amino acid into resin (1) H-Phe-O-Trt (2-Cl) -resin (179 mg) and DMF (4.5 mL) were added to the reaction vessel and allowed to swell for 20 minutes. After removing DMF, Fmoc-Asn (Trt) -OH 0.5M DMF solution (0.8 mL), DIC 0.2M DMF solution (2.0 mL), and Oxyma 0.5M DMF solution (0.8 mL) were added. After reacting by microwave (MW) irradiation at 75 ° C. for 5 minutes, the reaction solvent was removed. Washing with DMF (18 mL) gave Fmoc-Asn (Trt) -Phe-O-Trt (2-Cl) -resin.

2. Deprotection of Fmoc group (2) A 20% piperidine / DMF solution (4.5 mL) was added to Fmoc-Asn (Trt) -Phe-O-Trt (2-Cl) -resin. After stirring for 3 minutes, the reaction solvent was removed and 20% piperidine / DMF solution (4.5 mL) was added. After stirring for 10 minutes, the reaction solvent was removed and washed with DMF (18 mL) to obtain H-Asn (Trt) -Phe-O-Trt (2-Cl) -resin.

3. Coupling reaction (3) H-Asn (Trt) -Phe-O-Trt (2-Cl) -resin, Fmoc-His (Trt) -OH 0.5M DMF solution (0.8mL), DIC 0.5M DMF solution ( 0.8 mL) and Oxyma 0.2M DMF solution (2.0 mL) were added.
(4) After reacting by MW irradiation at 50 ° C. for 10 minutes, the reaction solvent was removed. The resin was washed with DMF (4.5 mL × 4 times) to obtain Fmoc-His (Trt) -Asn (Trt) -Phe-O-Trt (2-Cl) -resin.

(5) Thereafter, deprotection and Fmoc amino acid coupling were repeated according to the same procedures as in (2) to (4). (Coupling other than Fmoc-His (Trt) -OH was carried out by MW irradiation at 75 ° C. for 5 minutes).
(6) Fmoc-Glu (tBu) -OH at the 16th residue was coupled, followed by deprotection of the Fmoc group, and then the resin was washed with dichloromethane and MeOH and dried under reduced pressure. Got.
4). Cleavage of deprotected peptide from resin and purity measurement (7) 16-residue peptide-resin (partially collected approximately 20 mg) was added to TFA / TIS / H ₂ O / EDT (volume ratio TFA / TIS / H ₂ O /EDT=92.5/2.5/2.5/2.5) (2 mL) was added and stirred for 2 hours. After filtration and removing TFA, ethyl acetate and water were added for liquid separation. The aqueous layer was separated and washed by adding ethyl acetate (twice). The aqueous layer was diluted with purified water and analyzed by HPLC (HPLC purity 63.4%) (see FIG. 15 and Table 13, gradient program C).

[Experimental Example 8] B-fragment-resin (H-Lys (Boc) -His (Trt) -Leu-Asn (Trt) -Ser (tBu) -Met-Glu (tBu) -Arg (Pbf) -Val-Glu (tBu) -Trp (Boc) -Leu-Arg (Pbf) -Lys (Boc) -Lys (Boc) -Leu-Gln (Trt) -Asp (tBu) -Val-His (Trt) -Asn (Trt)- Synthesis of Phe-O-Trt (2-Cl) -resin) (side chain protected BFR-resin) (using M-Revo (registered trademark))

1. Introduction of Fmoc-amino acid into resin (1) Under argon atmosphere, Cl-Trt (2-Cl) -resin (10.0 g), Fmoc-Phe-OH (15.0 g), DIEA (6.75 mL), dichloroethane in a reaction vessel (100 mL) was added. After stirring for 3 hours or more using M-Revo (registered trademark), the reaction solvent was removed. DIEA (1.35 mL), MeOH (10 mL), and dichloromethane (70.0 mL) were added, and the mixture was stirred for 20 minutes using M-Revo (registered trademark) under an argon atmosphere. Washing with DMF, dichloromethane and DMF gave Fmoc-Phe-O-Trt (2-Cl) -resin.

2. Deprotection of Fmoc group (2) A 20% piperidine / DMF solution was added to Fmoc-Phe-O-Trt (2-Cl) -resin in a volume (about 10 v / w) that allowed stirring. After stirring for 20 minutes using M-Revo (registered trademark), the reaction solvent was removed. Washing with DMF, dichloromethane and DMF gave H-Phe-O-Trt (2-Cl) -resin.

3. Coupling reaction (3) Add Fmoc-Asn (Trt) -OH (23.1 g), HOBt (5.24 g), DIC (6.00 mL), DMF (70.0 mL) to the flask and use M-Revo (registered trademark) And stirred for 30 minutes or longer.
(4) The solution of (3) was added to H-Phe-O-Trt (2-Cl) -resin and stirred for 2 hours or more using M-Revo (registered trademark).
(5) The reaction solvent was removed, and the resin was washed with DMF, dichloromethane, and DMF to obtain Fmoc-Asn (Trt) -Phe-O-Trt (2-Cl) -resin.
(6) Thereafter, deprotection and coupling were repeated in the same manner as in (2) to (5).
Residue 10 (Fmoc-Arg (Pbf) -OH), Residue 15 (Fmoc-Arg (Pbf) -OH), Residue 17 (Fmoc-Met-OH), Residue 21 (Fmoc-His (Trt) -OH) and the 22nd residue (Fmoc-Lys (Boc) -OH) were subjected to double coupling. The 16th residue (Fmoc-Glu (tBu) -OH) was triple-coupled and the 19th residue (Fmoc-Arg (Pbf) -OH) was coupled 4 times.
(7) After coupling Fmoc-Lys (Boc) -OH at the 22nd residue, after deprotecting the Fmoc group according to the same method as (2), the resin was washed with dichloromethane and MeOH and dried under reduced pressure. A chain-protected BFR-resin (78.48 g) was obtained.
4). Cleavage of the protected peptide from the resin and purity measurement (8) Acetic acid / dichloromethane (volume ratio of acetic acid / dichloromethane = 1/9) was added to the side chain protected BFR-resin (partially collected approximately 20 mg) for 2 hours. Stir. The reaction solution was diluted with purified water / acetonitrile and then analyzed by HPLC (HPLC purity 92.0%) (see FIG. 16 and Table 14, gradient program D).

Comparative Example 4 B-fragment resin (H-Lys (Boc) -His (Trt) -Leu-Asn (Trt) -Ser (tBu) -Met-Glu (tBu) -Arg (Pbf) -Val-Glu (tBu) -Trp (Boc) -Leu-Arg (Pbf) -Lys (Boc) -Lys (Boc) -Leu-Gln (Trt) -Asp (tBu) -Val-His (Trt) -Asn (Trt)- Synthesis of Phe-O-Trt (2-Cl) -resin) (side chain protected BFR-resin) (using a shaker)

1. Introduction of Fmoc-amino acid into resin (1) Under argon atmosphere, Cl-Trt (2-Cl) -resin (2.00 g), Fmoc-Phe-OH (2.5 eq.), DIEA (2.5 eq.) In a reaction vessel Dichloroethane (about 10 v / w) was added. After stirring for 3 hours or more using a shaker, the reaction solvent was removed. DIEA (0.5 eq.), MeOH (2.0 mL) and dichloromethane (20 mL) were added, and the mixture was stirred for 20 minutes using a shaker. Washing with DMF (20 mL) gave Fmoc-Phe-O-Trt (2-Cl) -resin.

2. Deprotection of Fmoc group (2) A 20% piperidine / DMF solution (14 mL) was added to Fmoc-Phe-O-Trt (2-Cl) -resin. After stirring for 20 minutes using a shaker, the reaction solvent was removed. Washing with DMF, dichloromethane and DMF gave H-Phe-O-Trt (2-Cl) -resin.

3. Coupling reaction (3) Add Fmoc-Asn (Trt) -OH (2.5 eq.), HOBt (2.5 eq.), DIC (2.5 eq.), DMF (18.0 mL) to the flask, and use a shaker. And stirred for 30 minutes or longer.
(4) The solution of (3) was added to H-Phe-O-Trt (2-Cl) -resin and stirred for 2 hours or more using a shaker to remove the reaction solvent. The resin was washed with DMF (20.0 mL) to obtain Fmoc-Asn (Trt) -Phe-O-Trt (2-Cl) -resin.
(5) Thereafter, deprotection and Fmoc-amino acid coupling were repeated according to the same procedures as in (2) to (4).
(6) After coupling Fmoc-Lys (Boc) -OH at the 22nd residue, after deprotecting the Fmoc group according to the same method as (2), the resin was washed with MeOH (20 mL) and dried under reduced pressure. Then, a side chain protected BFR-resin (12.18 g) was obtained.

4). Cleavage of protected peptide from resin and purity measurement (7) Add degassed Clearage mixture (76 mL) to side chain protected BFR-resin (7.59 g) and use a shaker under argon atmosphere. Stir for hours.
(8) The resin was further washed with a clearance mixture (38 mL) and dichloromethane (76 mL).
(9) After adding hexane (760 mL) to the reaction solution and concentrating under reduced pressure, dichloromethane (15 mL) was added to dissolve the crystals. IPE (380 mL) was added to this solution to crystallize crystals, and the crystals were collected by pressure filtration.
(10) After drying the crystals under reduced pressure, the obtained side chain protected B-fragment crystals were dissolved in purified water / acetonitrile and measured by HPLC (HPLC purity 63.8%). As a result, the purity of the target B-fragment was 63.8% (t _R = 28.4), whereas the content of detrityl compound produced as a by-product was found to be 9.2% (t _R = 24.2). (See FIG. 17 and Table 15, Gradient program A).

[Comparative Example 5] B-fragment-resin (H-Lys (Boc) -His (Trt) -Leu-Asn (Trt) -Ser (tBu) -Met-Glu (tBu) -Arg (Pbf) -Val-Glu (tBu) -Trp (Boc) -Leu-Arg (Pbf) -Lys (Boc) -Lys (Boc) -Leu-Gln (Trt) -Asp (tBu) -Val-His (Trt) -Asn (Trt)- Phe-O-Trt (2-Cl) -resin) (side chain-protected BFR-resin) (using a fully automated microwave peptide synthesizer (Initiator + Alstra manufactured by Biotage Japan))

1. Introduction of Fmoc-amino acid into resin (1) H-Phe-O-Trt (2-Cl) -resin (118 mg) and DMF (4.5 mL) were added to a reaction vessel and allowed to swell for 20 minutes. After removing DMF, add Fmoc-Asn (Trt) -OH 0.5M DMF solution (0.8 mL), DIC 0.5M DMF solution (0.8 mL), HOBt 0.2M DMF solution (2.0 mL) After reacting by wave (MW) irradiation, the reaction solvent was removed. The resin was washed with DMF (18 mL) to obtain Fmoc-Asn (Trt) -Phe-O-Trt (2-Cl) -resin.

2. Deprotection of Fmoc group (2) Add 20% piperidine / DMF solution (4.5 mL) to Fmoc-Asn (Trt) -Phe-O-Trt (2-Cl) -resin and stir for 3 min. did. Further, 20% piperidine / DMF solution (4.5 mL) was added and stirred for 10 minutes, and then the reaction solvent was removed. The resin was washed with DMF (18 mL) to obtain H-Asn (Trt) -Phe-O-Trt (2-Cl) -resin.

3. Coupling reaction (3) Fmoc-His (Trt) -OH 0.5M DMF solution (0.8 mL), DIC 0.5M DMF solution (0.8 mL) in H-Asn (Trt) -Phe-O-Trt (2-Cl) -resin mL) and HOBt 0.2M DMF solution (2.0 mL).
(4) The whole amount was reacted by MW irradiation at 50 ° C. for 10 minutes, and then the reaction solvent was removed. The resin was washed with DMF (18 mL) to obtain Fmoc-His (Trt) -Asn (Trt) -Phe-O-Trt (2-Cl) -resin.
(5) Thereafter, deprotection and Fmoc-amino acid coupling were repeated following the same procedures as in (2) to (4) (Coupling other than Fmoc-His (Trt) -OH was irradiated with MW for 5 minutes at 75 ° C. Reaction).
(6) After coupling Fmoc-Lys (Boc) -OH at the 22nd residue, after deprotecting the Fmoc group according to the same method as (2), the resin was washed with dichloromethane and MeOH and dried under reduced pressure. A chain-protected BFR-resin was obtained.
4). Cleavage of the protected peptide from the resin and measurement of purity (7) A clearance mixture (2 mL) was added to a side chain protected BFR-resin (approximately 20 mg of a partially collected product) and stirred for 2 hours. The reaction solution was diluted with acetonitrile and analyzed by HPLC. As a result, the purity of the target B-fragment was found to be 75.5% (t _R = 20.4), whereas the content of detrityl compound produced as a by-product was 7.9% (t _R = 7.9). (See FIG. 18 and Table 16, Gradient program D).

The results of Experimental Examples 5 to 8 and Comparative Examples 3 to 5 are summarized in Table 17.

From the above results, it was confirmed that a large amount of highly purified long-chain peptide was obtained by the production method of the present invention. Moreover, it has confirmed that the production | generation of the intermediate fragment detrityl body in the peptide solid-phase synthesis method was reduced by the manufacturing method of this invention. That is, it was confirmed that the production method of the present invention can suppress a side reaction and can synthesize a large amount of a long-chain peptide with high purity.

[Experimental Example 9] Synthesis of pentaalanine (H-Ala-Ala-Ala-Ala-Ala-OH) (using M-Revo (registered trademark))
1. Introduction of Fmoc-amino acid into resin (1) Wang resin (paramethoxybenzyl alcohol resin, 5.0 g) and DMF (35 mL) were added to a reaction vessel to swell the resin. After removing DMF, Fmoc-Ala-OH 2.41 g (2.5 eq.), HOBt (1.05 g, 2.5 eq.), DMF (35 mL), and DIC (1.2 mL, 2.5 eq.) Were added.
(2) Using M-Revo (registered trademark), the mixture was centrifuged for 2 hours or more.
(3) The reaction solvent was removed, and the Fmoc-Ala-OH introduced resin was washed with DMF (35 mL), dichloromethane (35 mL), and DMF (35 mL) using M-Revo (registered trademark).
2. Deprotection of Fmoc group (4) 20% piperidine / DMF (35 mL) was added to the obtained Fmoc-Ala-Wang-resin.
(5) Using M-Revo (registered trademark), the mixture was centrifuged for 20 minutes or more.
(6) The reaction solvent was removed and washed with DMF (35 mL), dichloromethane (35 mL), and DMF (35 mL) using M-Revo (registered trademark).
(7) It was confirmed by the Kaiser test that the resin beads were colored.
(8) H-Ala-Wang-resin was obtained.

3. Fmoc-Ala-OH (2.41 g, 2.5 eq.), HOBt (1.05 g, 2.5 eq.), DMF (35 mL), DIC (1.2 mL, 2.5 eq) were added to the reaction vessel for the coupling reaction (9) (8). .) Was added.
(10) Using M-Revo (registered trademark), the mixture was centrifuged for 1 hour or more.
(11) The reaction solvent was removed, and the Fmoc-Ala-OH introduced resin was washed with DMF (35 mL), dichloromethane (35 mL), and DMF (35 mL) using M-Revo (registered trademark).
(12) A small amount of Fmoc-Ala-OH introduced resin was sampled, and it was confirmed by a Kaiser test that the resin beads were not colored. If the resin beads are colored by the Kaiser test, the operations (9) to (11) are repeated until the resin beads are not colored.
(13) Operations (4) to (12) were performed until Boc-Ala-Ala-Ala-Ala-Ala-Wang-resin was obtained. However, Boc-Ala-OH was introduced as the N-terminal amino acid.
(14) After coupling Boc-Ala-OH, wash with dichloromethane (35 mL) and MeOH (35 mL), dry under reduced pressure, and add the following Boc-Ala-Ala-Ala-Ala-Ala-Wang-resin Obtained.

4). Excision of peptide from resin (15) Boc-Ala-Ala-Ala-Ala-Ala-Wang-resin with TFA / TIS / H ₂ O (volume ratio TFA / TIS / H ₂ O = 95 / 2.5 / 2.5) ( 35 mL) was added and shaken for 2 hours.
(16) The reaction solution was concentrated under reduced pressure and crystallized with IPE (250 mL).
(17) The crystals were filtered under reduced pressure.
(18) It dried under reduced pressure at room temperature and obtained the following H-Ala-Ala-Ala-Ala-Ala-OH.TFA salt (604 mg).

[Comparative Example 6] Synthesis of pentaalanine (H-Ala-Ala-Ala-Ala-Ala-OH) (using a shaker)
1. Introduction of Fmoc-amino acid into resin (1) Wang-resin (0.50 g) and DMF (5 mL) were added to a reaction vessel to swell the resin. After removing DMF, Fmoc-Ala-OH (775 mg, 6 eq.), HOBt (337 mg, 6 eq.), DMF (2.5 mL), and DIC (386 μL, 6 eq.) Were added.
(2) Shake for more than 2 hours using a shaker.
(3) The reaction solvent was removed, and the Fmoc-Ala-OH introduced resin was washed with DMF (5 mL), dichloromethane (5 mL), and DMF (5 mL) using a shaker.

2. Deprotection of Fmoc group (4) 20% piperidine / DMF (5.5 mL) was added to the resulting Fmoc-Ala-Wang-resin.
(5) Using a shaker, shake for 20 minutes or more.
(6) The reaction solvent was removed, and the mixture was washed with DMF (5 mL), dichloromethane (5 mL), and DMF (5 mL) using a shaker.
(7) It was confirmed by the Kaiser test that the resin beads were colored.
(8) H-Ala-Wang-resin was obtained.

3. Fmoc-Ala-OH (386 mg, 2.5 eq.), HOBt (168 mg, 2.5 eq.), DMF (5.5 mL), DIC (206 μL, 2.5 eq) were added to the reaction vessel for the coupling reaction (9) (8). .) Was added.
(10) Using a shaker, shake for 1 hour or more.
(11) The reaction solvent was removed, and the Fmoc-Ala-OH introduced resin was washed with DMF (5 mL), dichloromethane (5 mL), and DMF (5 mL) using a shaker.
(12) A small amount of Fmoc-Ala-OH introduced resin was sampled, and it was confirmed by a Kaiser test that the resin beads were not colored. If the resin beads are colored by the Kaiser test, the operations (9) to (11) are repeated until the resin beads are not colored.
(13) Operations (4) to (12) were performed until Boc-Ala-Ala-Ala-Ala-Ala-Wang-resin was obtained. However, Boc-Ala-OH was used as the protected amino acid (N-terminal amino acid) used in the final coupling reaction.
(14) After coupling with Boc-Ala-OH, washed with dichloromethane (5 mL), MeOH (5 mL), and dried under reduced pressure. Boc-Ala-Ala-Ala-Ala-Ala-Wang-resin (0.68 g) Got.

4). Cutting out deprotected peptide from resin (15) TFA / TIS / H ₂ O (volume ratio TFA / TIS / H ₂ O = 95 / 2.5 / 2.5) to Boc-Ala-Ala-Ala-Ala-Ala-Wang-resin ) (5 mL) was added and shaken for 2 hours.
(16) After removing the resin by filtration, the reaction solution was concentrated under reduced pressure, and IPE (25 mL) was added to the residue for crystallization.
(17) The crystals were filtered under reduced pressure.
(18) It dried under reduced pressure at room temperature and obtained H-Ala-Ala-Ala-Ala-Ala-OH * TFA salt (60.7 mg).

[HPLC analysis (Experimental Example 9 and Comparative Example 6)]
The results of HPLC analysis of acetonitrile solutions of the products of Experimental Example 9 and Comparative Example 6 are shown in Table 17 and FIGS. Table 17 shows the retention time and content of the product (pentaalanine) in the HPLC analysis of the acetonitrile solutions of the products of Experimental Example 9 and Comparative Example 6. 19 and 20 show HPLC chromatograms of acetonitrile solutions of the products of Experimental Example 9 and Comparative Example 6, respectively. This HPLC analysis was performed under the following HPCL conditions.

HPLC condition column: Waters X Bridge
Mobile phase: 0.1% TFA aqueous solution Analysis time: Approx. 10 minutes Flow rate: 1 mL / min
Detector: UV 220 nm

From the above results, it was confirmed that a large amount of highly purified long-chain peptide was obtained by the production method of the present invention.

The production method of the present invention is useful for the production of peptides. In particular, it is useful for mass synthesis of long-chain peptides while suppressing side reactions.

DESCRIPTION OF SYMBOLS 1 Rotating body for stirring 5 Stirrer main body 10 Main body 11 Rotation drive shaft 12 Suction port 13 Cylindrical rotating member 13A Top plate 13B Bottom plate 14 Discharge port 16 Flow path 18 Connection portion 20 Drive shaft 21 Cylindrical housing 22A-22D Release opening 23 Suction Tube portion 24A to 24D Extrusion protruding plate portion 25A to 25D Suction opening 30 Communication hole 38 Stirrer 40 Stirring rotating body 41 Stirring rotating body main body 41a Stirring rotating body main body 41a A substantially circular upper surface 41b Stirring rotating body 41b A substantially circular bottom surface of the main body 41c A side surface which is the outer peripheral surface of the main body of the stirring rotor 42 A suction port of the stirring rotor 44 A discharge port of the stirring rotor 46 A flow path of the stirring rotor 48 A connection 50 A flow resistance Body 51 Main body of flow resistor 51a Upper surface of substantially circular shape of main body of flow resistor 51b Substantially circular bottom surface of main body of flow resistor 51c Main body of flow resistor Side surface 52 which is an outer peripheral surface 52 Flow resistor suction port 54 Flow resistor discharge port 56 Flow resistor flow path 60 Drive shaft a Rotation drive shaft rotation direction b Cylindrical rotation member rotation direction d1, d4 External discharge flow e1 ~ E3 Suction flow g1 ~ g4, h1 ~ h4 Suction flow C, L Center axis

Claims (5)

A method for producing a peptide, characterized in that the peptide is solid-phase synthesized under stirring of a centrifugal stirring body without blades. A centrifugal stirring body without blades
A main body that rotates about a rotation axis;
An inlet provided on the surface of the body;
A discharge port provided on the surface of the main body;
A flow path connecting the suction port and the discharge port,
The suction port is disposed at a position closer to the rotation shaft than the discharge port,
The manufacturing method according to claim 1, wherein the discharge port is a rotating body for stirring, which is disposed at a position on the outer side in the centrifugal direction from the rotation shaft than the suction port. Use of a centrifugal stirring body without blades for peptide synthesis by the solid phase method. A reaction vessel for peptide solid phase synthesis equipped with a centrifugal stirring body without blades. The reaction vessel for peptide solid phase synthesis according to claim 4, comprising a glass filter.

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